Fig. 2

Heterologously expressed SLC35B1 and SLC35B1/Isoform 2 are ATP and ADP carriers. The indicated SLC35B1 variants (labeled by asterisk in a, which were described in the legend to Fig. 1a, plus a phosphomimetic variant of Isoform 2 with aspartate residues at positions 15 and 29 instead of serines (termed Isoform 2DD, lane 5), and the GFP-tagged version of SLC35B1 were expressed in different E. coli cells. a, b Membranes were isolated from the indicated E. coli cells, and samples (25 µg protein) analyzed by SDS-PAGE and Western blotting using an SLC35B1-specific antibody, which was characterized in Supplementary Fig. 1a (a). Non-induced cells served as a negative control and are shown in lane 1. Coomassie staining the blot for total protein served as a loading control (b). A molecular mass standard (M) was run in parallel and electronically copied from the stained blot to the Western blot image. c–f Uptake of 50 µM [α³²P]ATP (c–e) or [α³²P]ADP (d–f) into SLC35B1-expressing (c, d) or SLC35B1/Isoform 2-expressing cells (e, f) was measured and compared to uptake by non-induced cells. Data are reported as the mean of at least three independent experiments and are shown with the standard error of the mean (SEM). Related substrate saturation experiments and corresponding Eadie-Hofstee analyses revealed apparent K_M values for ATP and ADP plus maximal uptake rates and are shown in Supplementary Fig. 4